Single aperture confocal scanning epi-illumination microscope

Abstract

A single aperture confocal scanning epi-illumination microscope comprises an assembly including, in one embodiment, a pair of intermediate lenses to create a second field plane conjugate to the specimen plane in both the incident and reflected light paths, with a single aperture positioned at this second conjugate field plane and controllably scanned through the plane to create the incident light beam as well as to mask the returning light before viewing. In a second embodiment, only a single lens is included in the assembly and the objective lens may be positioned at its correct tube length, a distance substantially shorter, or a non-standard objective lens may be used.

What Is Claimed Is: 
     
       1. A confocal scanning microscope for viewing a specimen with epi-illumination, the microscope including a light source for creating incident light to illuminate the specimen, first optic means for focusing the incident light to form a specimen plane, means to separate incident light from light returning from the specimen, the specimen plane being adapted for receiving the specimen desire be viewed, an aperture means, a second optic means to create a field plane conjugate to the specimen plane for incident light, said second optic means being positioned between the light separating means and the aperture means, said first optic means having means to create a field plane conjugate to the specimen plane for light returning from the specimen, said first optic means being positioned between the aperture means and the specimen, said conjugate field planes being coincident with the aperture means being positioned thereat, and means to create a conjugate field plane for returning light whereat the specimen may be viewed. 
     
     
       2. The device of claim 1 wherein the first and second optic means each comprise standard objective lenses. 
     
     
       3. The device of claim 2 wherein the first optic means also has means to act as the primary objective of the microscope, there being no other optical element positioned between the aperture means and the specimen for magnification of the specimen. 
     
     
       4. The device of claim 3 wherein the first optic means has a tube length, and the microscope further comprises means to position said first optic means at a distance substantially equal to its tube length from the aperture means. 
     
     
       5. The device of claim 3 wherein the first optic means has a tube length, and the microscope further comprises means to position said first optic means at a distance from the aperture means substantially less than its tube length. 
     
     
       6. The device of claim 1 wherein the first optic means also has means to act as the primary objective of the microscope, there being no other optical element positioned between the aperture means and the specimen for magnification of the specimen. 
     
     
       7. The device of claim 6 wherein the first optic means has a tube length, and the microscope further comprises means to position said first optic means at a distance substantially equal to its tube length from the aperture means. 
     
     
       8. The device of claim 1 wherein the first optic means comprises a pair of lenses, at least one said lenses being a standard objective lens. 
     
     
       9. The device of claim 8 wherein the first optic means lens pair comprises two standard objective lenses. 
     
     
       10. The device of claim 9 wherein the second optic means comprises a single standard objective lens. 
     
     
       11. The device of claim 1 wherein the aperture means is substantially smaller in cross-sectional area than that portion of the specimen being viewed, and further comprising means to scan that portion of the specimen being viewed with incident light. 
     
     
       12. The device of claim 11 wherein the scanning means comprises means to move the aperture means within the first conjugate field plane. 
     
     
       13. The device of claim 12 wherein the aperture means comprises a single opening, and the scanning means comprises means to move said single opening within the first conjugate field plane. 
     
     
       14. The device of claim 12 wherein the aperture means comprises a disc, said disc having means defining a plurality of holes, and the scanning means comprises means to rotate the disc. 
     
     
       15. The device of claim 12 wherein the aperture means comprises a slit, and the scanning means comprises means to sweep or rotate said slit through the first conjugate field plane. 
     
     
       16. The device of claim 1 further comprising means to locate the co-incident conjugate field plane apart from an axis defined by the light traversing the space between the specimen plane and the light separating means. 
     
     
       17. The device of claim 16 wherein the conjugate field plane locating means comprises a plurality of mirrors, at least some of said mirrors being positioned in the optical axis. 
     
     
       18. The device of claim 17 wherein the conjugate field plane locating means comprises means to increase the accessibility of the conjugate field 
     
     
       19. In a confocal scanning microscope for viewing a specimen with epi-illumination, the microscope including a light source for creating incident light to illuminate the specimen, means for focusing the light to form a specimen plane, means to separate incident light from light reflected from the specimen, the specimen plane being adapted for receiving the specimen desired to be viewed, and an aperture means, the improvement comprising two lenses to create a field plane conjugate to the specimen plane for both incident light and light returning from the specimen, one of said lenses being positioned between the light separating means and the aperture means, and the other of said lenses being positioned between the aperture means and the specimen, both of said lenses being thereby positioned in both the incident light path and the reflected light path, the aperture means being positioned at the conjugate field plane, and means to create a second conjugate field plane for returning light whereat the specimen may be viewed. 
     
     
       20. The device of claim 19 wherein the aperture means is substantially smaller in cross-sectional areas than that of the specimen, and further comprising means to scan the specimen with incident light. 
     
     
       21. The device of claim 20 wherein the scanning means comprises means to move the aperture means within the first conjugate field plane. 
     
     
       22. The device of claim 21 wherein the aperture means comprises a single opening, and the scanning means comprises means to move said single opening within the first conjugate field plane. 
     
     
       23. The device of claim 21 wherein the aperture means comprises a disc, said disc having means defining a plurality of holes, and the scanning means comprises means to rotate the disc. 
     
     
       24. The device of claim 21 wherein the aperture means comprises a slit, and the scanning means comprises means to sweep or rotate said slit through the first conjugate field plane. 
     
     
       25. The device of claim 19 further comprising means to locate the first conjugate field plane apart from an optical axis defined by the light traversing the space between the specimens plane and the light separating means. 
     
     
       26. The device of claim 25 wherein the conjugate field plane locating means comprises a plurality of mirrors, at least some of said mirrors being positioned in the optical axis. 
     
     
       27. The device of claim 26 wherein the conjugate field plane locating means comprises means to increase the accessibility of the conjugate field plane. 
     
     
       28. The device of claim 19 further comprising means to change the magnification of the microscope by changing the second lens. 
     
     
       29. In a confocal scanning microscope for viewing a specimen, the microscope having means for focusing incident light at the specimen to form a specimen plane thereat, an aperture means, and means for separating light reflected from the specimen from incident light, the improvement comprising two lenses to create a field plane in both the incident and reflected light paths conjugate to the specimen plane, said aperture means being positioned at said conjugate field plane, one of said two lenses being positioned between the light separating means and the aperture means, and the other of said two lenses being positioned between the aperture means and the specimen plane so that they both are traversed by both incident and reflected light. 
     
     
       30. The device of claim 29 further comprising means to induce a field plane above the light separating means whereat the specimen may be viewed, said field plane being conjugate to the specimen plane. 
     
     
       31. The device of claim 29 further comprising means to scan the incident light across the specimen to create a real time image of said specimen. 
     
     
       32. The device of claim 29 further comprising means to scan the incident light across the specimen to thereby create a plurality of images, means to record the plurality of images, and means to assimilate said plurality of images into a single composite image of the specimen. 
     
     
       33. The device of claim 29 wherein the light traversing the path between the field plane and the specimen plane form an optical axis and further comprising means to divert said light from the optical axis to thereby locate the field plane off of the optical axis. 
     
     
       34. The device of claim 33 wherein the light diverting means comprises two pairs of mirrors.